1. Field of the Invention
The present invention relates to purified Mullerian Inhibiting Substance, methods for its purification and its use in the inhibition of tumor growth.
2. Description of the Prior Art
A substance with the properties of inducing regression of the Mullerian duct, the fetal analog of the uterus, fallopian tube and vagina, was suspected in 1916 when Lillie (Science 43:611-613 (1916)) described the freemartin calf, an abnormal female product of heterosexual twinning, long known to farmers and veterinarians. Immunologists soon appreciated the chimeric characteristics of this "experiment in nature", and endocrinologists began to understand the important developmental implications of this masculinized female.
Jost, A. (CR Soc. Biol. 140:460, 463 (1946), ibid 141:135 (1947)), experimentally reproduced selected freemartin effects in embryo rabbits at a stage when they were sexually indifferent, by removing gonads and replacing them with either testis, ovary, no gonad, or crystalline testosterone. Those replaced with ovary or no gonad developed normal Mullerian ducts which later formed uterus, fallopian tubes and vagina. Those replaced with testis developed Wolffian ducts which later formed vas, seminal vesicals, and epididymis, while the Mullerian ducts regressed. Those replaced with testosterone alone stimulated the Wolffian duct structures but did not regress the Mullerian duct. These results caused Jost to speculate the existence of a testicular product which caused regression of the Mullerian duct, coining the term "Mullerian Inhibiting Substance" (MIS).
Donahoe et al (J. Surg. Res. 23:141-148 (1977)) demonstrated that a high level of MIS persists in newborn calf testis for up to 8 weeks after birth. This tissue has provided a ready source for the partial purification and characterization of the biologically active moiety by guanidine hydrochloride extraction (see for example Swann, D. A. et al, Developmental Biology 69:73-84 (1979), Donahoe et al, Pediatric Andrology 37-46, 1981 Martinus Nijhoff Publishers, The Hague/Boston/London, and Donahoe et al, Science:205, 913-915 (1979)).
Purification of MIS from calf testis has been a tedious undertaking since large quantities of material are consumed in the vital assay required for activity confirmation. In spite of these problems, interest in MIS purification remains high since impure fractions with MIS activity are cytotoxic to human ovarian cancer in vitro (Donahoe, P. K., et al Science 205:913-915 (1979)).
Incubation of fetal calf testis in media for 4 hours showed that MIS can also be secreted (Josso, N. et al, Biol. Reprod. 13:163-167 (1975)). Homogenization of testis tissue, however, has not yielded active preparations (Donahoe, P. K. et al Cryobiol. 14:534-542 (1977), Josso, N. et al, Recent Prog. Hormone Res. 33:117-167 (1977)).
The aforementioned previous attempts at the purification of MIS yielded preparations with only approximate 30-fold enhancement in purity. Furthermore, the use of guanidine hydrochloride as a disassociative solvent in the extraction of testicular tissue, is a method which although instructive, requires a subsequent lengthy cesium chloride sedimentation prior to further fractionation.
Because of the great applicability of purified MIS, and the need to streamline the process of purification, there continues to exist a need for an efficient method of purification for, and highly purified fractions of this material.